Summary Over 40 million anesthetics are administered for surgical procedures in the United States every year. During these anesthetics, many patients receive halogenated anesthetic drugs such as isoflurane, sevoflurane, or desflurane. However, the pharmacologic mechanisms by which these drugs cause anesthesia are unclear. A detailed understanding of these mechanisms may foster development of safer, more selective anesthetics with fewer undesired side effects (e.g., cardiorespiratory depression and cognitive dysfunction), and drugs that facilitate recovery from general anesthesia. Using methods in yeast molecular biology, electrophysiology, and biochemistry as well as rodent studies, we will test the hypothesis that halogenated anesthetics cause anesthesia in part by direct binding and activation of TASK-3 potassium channels. TASK-3 is a membrane associated tandem pore potassium channel protein expressed in the brain and spinal cord that regulates neuronal excitability. TASK-3 potassium channel function is activated by isoflurane, sevoflurane, desflurane, and other halogenated anesthetics; and knockout mice lacking the TASK-3 gene require significantly higher doses of halogenated anesthetics for induction of loss of righting and to maintain immobility during a noxious stimulus. In Aim 1, we will use a yeast-based, high throughput screen of 4,693 TASK-3 missense mutants to identify TASK-3 amino acid residues critical for its function and for halogenated anesthetic activation. In Aim 2, we will purify functional wild-type and anesthetic-resistant mutant TASK-3 protein and measure their single channel function at baseline and following halogenated anesthetic treatment using the lipid bilayer voltage clamp method. Finally, in Aim 3 will administer potent and selective TASK-3 antagonist compounds to isoflurane anesthetized rats. We will quantify the TASK-3 antagonists' effects on the rat electroencephalogram (EEG) and on behavioral end points of anesthesia, loss of righting reflex and immobility during a noxious stimulus. The above studies will clarify the mechanism by which anesthetics interact with TASK-3 channels to cause activation. They will also reveal the contribution of TASK-3 to isoflurane anesthesia in a live, genetically unmodified animal.